PROJECT SUMMARY The goal of this supplement request is to purchase a compact mass spectrometer (CMS) for the bromodomain biophysical and drug discovery studies covered by the R35 GM128840 award. Bromodomains bind acyl- lysines on histones. The role of bromodomain-regulated transcription in human disease is well appreciated with bromodomain inhibitors in clinical trials. Despite these achievements, several critical questions remain. For example, bromodomains are localized disproportionately at super-enhancers. The basis of this localization is unknown but important given that super-enhancers are enriched at loci with oncogenic potential. We hypothesize that tandem bromodomains act as a scaffold for acetylation-dependent chromatin reorganization; for instance, joining promotors with enhancers to drive transcription (Focus 1). We are taking a structural and biophysical approach to investigate the role of tandem bromodomains in maintaining chromatin conformations. We also hypothesize that metabolic changes induce post-translational modifications on histones that are ?read? by bromodomains. Yet, the acylation and protein binding specificity of bromodomains are poorly understood. To address this metabolic question, we are using biophysical, structural biology, and proteomic techniques to investigate bromodomain acylation selectivity and link acyl-CoA metabolism with transcription (Focus 2). In support of Focus 1 and 2, the requested CMS will include an electrospray ionization (ESI) source, an Open Port Sampling Interface (OPSI), and software to allow mass spectral analyses of acylated semisynthetic histones (Focus 1) and synthesized peptides (Focus 2). This CMS offers an attractive complement to the tools currently employed because of its ability to collect data in minutes in a walk-up manner, its installation in proximity to our peptide synthesis instrument, and lack of user fees. To aid mechanistic inquiries, we are developing inhibitors of bromodomains using a novel fragment-based NMR screening strategy with a current focus on the PBRM1 bromodomains (Focus 3). These chemical tools will distinguish the differential activities of bromodomains in disease models and lead to therapeutics targeting the PBRM1 axis in cancer. In support of Focus 3, the requested CMS will contain an APCI source along with automated TLC and ASAP sample introduction methods, which offers mass spectral data collection in seconds in a walk-up manner and with minimal sample preparation. The CMS instrument will be installed in the PIs synthetic chemistry laboratory as part of the Program in Chemical Biology (PCB) at the Medical College of Wisconsin. The PCB is equipped with the necessary infrastructure for the installation and operation of this instrument. The instrument will be maintained by PhD-level staff with experience in the operation and maintenance of similar instruments. Consistent with its record of major investments in biophysical research infrastructure and facilities, MCW has committed space to house the requested instrument, 50% of costs for its maintenance, and salary support for training and maintenance.